JP2022546777A - ANTI-VEGF SINGLE DOMAIN ANTIBODY AND APPLICATION THEREOF - Google Patents

Abstract

The present invention provides anti-VEGF single domain antibodies and applications thereof. Specifically, the present invention provides anti-VEGF single domain antibodies and their VHH chains. The invention further provides coding sequences encoding said single domain antibodies or VHH chains thereof, corresponding expression vectors and host cells capable of expressing said single domain antibodies, and methods of producing single domain antibodies of the invention. . The single domain antibody of the present invention can specifically discriminate human VEGFA, does not cross-react with VEGFB, VEGFC, VEGFD and has good specificity, and the single domain antibody of the present invention is capable of identifying human , mouse, rabbit, and monkey VEGFA simultaneously, can effectively block the interaction between VEGFA and VEGFR2, and between VEGFA and VEGFR1, and is highly potent against angiogenesis. Having an inhibitory effect, the single domain antibodies of the invention show better stability under non-formulated conditions.
[Selection figure] None

Description

The present invention relates to the technical field of biomedical or biopharmaceuticals, and more specifically to anti-VEGF single domain antibodies and their applications.

Existing vascular endothelial growth factor drugs include Bevacizumab (trade name is Avastin), Conbercept, Aflibercept and others. On February 13, 2018, the China State Food and Drug Administration (CFDA) approved eylea (aflibercept intraocular injection) for the treatment of diabetic macular edema in adults. The price of bevacizumab (bevacizumab, trade name is Avastin) for anti-tumor therapy is 1998 yuan (China) per bottle of 4 ml (containing 100 mg of bevacizumab), and the recommended dose is 5 mg/kg. , once every 14 days, the patient's weight is 60 kg, 300 mg of bevacizumab each time, that is, 3 doses twice a month, that is, 11988 yuan a month. The price of Conversecept for the treatment of eye diseases is 5550 yuan per bottle of 0.2ml (containing 10mg of Conversecept), and its dosing regimen is once a month for the first three months, then once a month. Once a month for three months, a total of six injections a year, that is, 33,300 yuan a year. In addition, the price of aflibercept is 5850 yuan per bottle of 0.1ml (containing 4mg of aflibercept), and its dosing plan is once every two months, six times a year, that is, one year. to 35,100 yuan.

Single domain antibody (nanobody, Nb), i.e. heavy chain single domain antibody VHH (variable domain of heavy chain of heavy-chain antibody) - a heavy-chain antibody that naturally lacks a light chain in the camel's body , HCAb) exist and consist of a single heavy chain variable region obtained by cloning the variable region, which is the smallest fully functional, stable antigen-binding unit currently available. . Single domain antibodies are characterized by high stability, high water solubility, easy humanization, high targeting and high penetrability, and have unimaginable roles in immunological experiments, diagnostics and therapeutics. Fulfill. Single domain antibodies are slowly becoming a new force in the new generation of antibody diagnostics and therapeutics.

To develop a new class of anti-VEGF single-domain antibodies with better specificity, blocking activity, better clinical efficacy, easier production, reduced production costs, and reduced patient medication burden It is an issue that urgently needs to be resolved.

An object of the present invention is to provide a new kind of anti-VEGF single domain antibody and its application.

Specifically, the object of the present invention is to effectively block the interaction between VEGFA and VEGFR2, and between VEGFA and VEGFR1, have a good inhibitory effect on angiogenesis and against solid tumors. It is an object of the present invention to provide a single domain antibody with good inhibitory effect and better specificity.

A first aspect of the invention provides a complementarity determining region (CDR) of an anti-VEGF single domain antibody VHH chain, wherein the complementarity determining region CDR of said VHH chain comprises SEQ ID NO. CDR1 shown in :1, SEQ ID NO. :2, and SEQ ID NO. : contains CDR3 shown in 3.

In another preferred example, said CDR1, CDR2 and CDR3 are separated by framework regions FR1, FR2, FR3 and FR4.

A second aspect of the invention provides a VHH chain of an anti-VEGF single domain antibody, said VHH chain comprising framework regions FR and complementarity determining regions CDRs according to the first aspect of the invention.

In another preferred example, the framework region FR is
(a) SEQ ID NO. FR1 shown in :4, SEQ ID NO. : FR2, SEQ ID NO. :6, and SEQ ID NO. :7, or (b) SEQ ID NO. FR1 shown in :10, SEQ ID NO. FR2 shown in :11, SEQ ID NO. : 12, and SEQ ID NO. : FR4 shown in 13.

In another preferred example, the VHH chain of said anti-VEGF single domain antibody has SEQ ID NO. :8 or 14.

Further provided is a heavy chain variable region of the novel anti-VEGF single domain antibody, said heavy chain variable region having SEQ ID NO. CDR1 shown in :1, SEQ ID NO. :2, and SEQ ID NO. : contains CDR3 shown in 3.

A third aspect of the invention provides an anti-VEGF antibody, said anti-VEGF antibody having a VHH chain according to the second aspect of the invention.

In another preferred example, said anti-VEGF antibody comprises a double chain antibody, a single chain antibody, a single domain antibody.

In another preferred example, said anti-VEGF antibody is selected from animal-derived antibody, chimeric antibody, humanized antibody, more preferably humanized antibody, human-animal chimeric antibody, more preferably fully humanized antibody.

In another preferred example, said anti-VEGF antibodies are antibody fragments such as Fab, Fab', (Fab')2 or other antibody derivatives known in the art, and IgA, IgD, IgE, IgG and IgM antibodies or other any one or more of the subtypes of the antibody.

In another preferred example, said anti-VEGF antibody is an anti-VEGF single domain antibody.

In another preferred example, said anti-VEGF antibody comprises monomers, bivalents (bivalent antibodies) and/or multivalents (multivalent antibodies).

In another preferred example, said anti-VEGF antibody has SEQ ID NO. : 8 or SEQ ID NO. :14.

In another preferred example, the VHH chain sequence of said anti-VEGF antibody is SEQ ID NO. :8 and/or SEQ ID NO. :14.

In another preferred example, said anti-VEGF antibody has SEQ ID NO. : 8 or SEQ ID NO. It contains two VHH chains with amino acid sequences as shown in:14.

In another preferred example, said anti-VEGF antibody has SEQ ID NO. :8 and/or SEQ ID NO. :14 and has a VHH chain with the amino acid sequence shown in FIG.

In another preferred example, the SEQ ID NO. :14 are connected by a linker between the two VHH chains.

In a preferred embodiment of the invention, said linker is selected from the sequence (GaSb) _x- ( _GmSn ) _y , where a, _b , _m , _n , x, y=0 or 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 (preferably a=4 and b=1, m=3 and n=1).

In a preferred embodiment of the invention, said linker is selected from the group consisting of GGGGSGGGGS (SEQ ID NO.:18), GS (SEQ ID NO.:19), GGGGS (SEQ ID NO.:20).

In another preferred example, the amino acid sequence of said anti-VEGF antibody is SEQ ID NO. :16.

In a preferred embodiment of the invention said bivalent anti-VEGF antibody is hubi-Nb24(Y).

A fourth aspect of the invention provides a polynucleotide, said polynucleotide being the CDR region of the anti-VEGF single domain antibody VHH chain according to the first aspect of the invention, encoding a protein selected from the group consisting of the VHH chain of an anti-VEGF single domain antibody as described or an anti-VEGF single domain antibody as described in the third aspect of the invention.

In another preferred example, the polynucleotide has SEQ ID NO. :9 or 15.

In another preferred example, the polynucleotide has SEQ ID NO. :17.

In another preferred example, said polynucleotide comprises DNA or RNA.

A fifth aspect of the invention provides an expression vector, said expression vector comprising a polynucleotide according to the fourth aspect of the invention.

In another preferred example, said expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

Preferably, said expression vector comprises a viral vector such as a Lentivirus, Adenovirus, AAV virus, Retrovirus, or a combination thereof.

A sixth aspect of the invention provides a host cell, said host cell comprising an expression vector according to the fifth aspect of the invention or whose genome comprises a gene according to the fourth aspect of the invention. of polynucleotides are incorporated.

In another preferred example, said host cell comprises a prokaryotic or eukaryotic cell.

In another preferred example, said host cell is selected from the group consisting of E. coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

In another preferred example, said prokaryotic cell is selected from the group consisting of E. coli, Bacillus subtilis, lactobacillus, streptomyces, proteus mirabilis, or combinations thereof.

In another preferred example, said eukaryotic cell is selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces cerevisiae, trichoderma, or a combination thereof. be done.

In another preferred example, said eukaryotic cells are from the group consisting of insect cells such as fall armyworm, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or combinations thereof. selected.

In another preferred example, said host cells are preferably mammalian cells, more preferably HEK293 cells, CHO cells, BHK cells, NSO cells or COS cells.

In another preferred example, said host cell is Pichia pastoris.

A seventh aspect of the present invention is
(a) obtaining a culture comprising said anti-VEGF single domain antibody by culturing the host cell according to the sixth aspect of the invention under conditions suitable for the production of single domain antibodies;
(b) isolating or recovering said anti-VEGF single domain antibody from said culture; and (c) optionally purifying and/or modifying to obtain the VEGF single domain antibody of step (b). and a method of producing an anti-VEGF single domain antibody.

In another preferred example, said anti-VEGF single domain antibody has SEQ ID NO. :8 or 14.

In another preferred example, said anti-VEGF single domain antibody has SEQ ID NO. :16.

An eighth aspect of the invention provides an immunoconjugate comprising:
(a) a VHH chain of an anti-VEGF single domain antibody according to the second aspect of the invention or an anti-VEGF single domain antibody according to the third aspect of the invention, and (b) a detectable marker , drugs, toxins, cytokines, radionuclides, enzymes, gold nanoparticles/nanorods, magnetic nanoparticles, viral coat proteins or VLPs, or combinations thereof.

In another preferred example, the radionuclide is
(i) Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177 , Re-188, or combinations thereof, and/or (ii) Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb- 212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe- 133, Yb-169, Yb-177, or a combination thereof.

In another preferred example, said coupling moiety is a drug or toxin.

In another preferred example, said drug is a cytotoxic drug.

In another preferred embodiment, the cytotoxic drug is an antitubulin drug, a DNA minor groove binding reagent, a DNA replication inhibitor, an alkylating reagent, an antibiotic, an antifolate, an antimetabolite, a chemosensitizer, a topoisomerase inhibitor. selected from the group consisting of a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.

In another preferred embodiment, examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding reagents, DNA alkylating reagents, and tubulin inhibitors; (auristatins), camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g. DM1 and DM4), taxanes, benzodiazepines or benzodiazepines benzodiazepine containing drugs (e.g. pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof .

In another preferred example, the toxin is auristatin (e.g., auristatin E, auristatin F, MMAE and MMAF), chlortetracycline, maytansyloid, gatexin, gatexin A-chain, combretastatin, docarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vincristine, vincristine colchicine, dihydroxyanthraxdione, actinomycin, diphtheria toxin, pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, volkensin A chain , alpha-sarcinus, gelonin, mitogellin, restrictocin, phenomycin, enomycin, curcin, croton, calicheamicin, Saponaria officinalis inhibitor, selected from the group consisting of glucocorticoids, or combinations thereof.

In another preferred example, said coupling moiety is a detectable marker.

In another preferred example, the coupling moiety is a fluorescent or luminescent marker, a radioactive marker, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product, a radionuclide, Biological toxins, cytokines (e.g. IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, magnetic nanoparticles, prodrug activating enzymes (e.g. , DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (eg cisplatin) or nanoparticles of any form.

In another preferred embodiment, said immunoconjugate is a multivalent (e.g. bivalent) VHH chain of an anti-VEGF single domain antibody according to the second aspect of the invention, according to the third aspect of the invention. of anti-VEGF single domain antibodies.

In another preferred example, said multivalent means that the amino acid sequence of said immunoconjugate is the VHH chain of the anti-VEGF single domain antibody according to the second aspect of the invention, according to the third aspect of the invention. of anti-VEGF single domain antibodies.

A ninth aspect of the present invention is
(a) a drug for inhibiting angiogenesis;
(b) the VHH chain of the anti-VEGF single domain antibody according to the second aspect of the invention, for use in the preparation of a medicament for treating a disease or condition associated with VEGF, the third aspect of the invention; Use of the anti-VEGF single domain antibody described in .

A tenth aspect of the invention provides a pharmaceutical composition, said pharmaceutical composition comprising:
(i) the complementarity determining region (CDR) of the anti-VEGF single domain antibody VHH chain according to the first aspect of the invention, the VHH chain of the anti-VEGF single domain antibody according to the second aspect of the invention; an anti-VEGF single domain antibody according to the third aspect of the invention or an immunoconjugate according to the ninth aspect of the invention; and (ii) a pharmaceutically acceptable vector.

In another preferred example, the coupling portion of said immunoconjugate is a drug, toxin, and/or therapeutic isotope.

In another preferred example, the pharmaceutical composition further comprises other drugs that treat tumors, such as cytotoxic drugs.

In another preferred example, said pharmaceutical composition is used to block the interaction between VEGFA and VEGFR2 and between VEGFA and VEGFR1.

In another preferred example, said pharmaceutical composition is in an injectable dosage form.

In another preferred example, said pharmaceutical composition is used for the preparation of a medicament for treating a disease or condition associated with VEGF, wherein said disease or condition is a tumor or cancer or an eye disease.

In another preferred example, the tumor or cancer comprises one or more of breast cancer, lung cancer, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, thyroid cancer, nasopharyngeal cancer, Not limited.

In another preferred example, the eye disease includes age-related macular degeneration, diabetic retinopathy, retinal vein occlusion, pathological myopia, neovascular glaucoma and other eye diseases involving neovascularization, including but not limited to: Not limited.

An eleventh aspect of the present invention is
(a) drugs for inhibiting angiogenesis;
(b) drugs for treating diseases or conditions associated with VEGF;
(c) for detection of human VEGF molecules,
(d) for flow detection;
(e) for cellular immunofluorescence detection,
(f) for tumor therapy;
(g) use of one or more of the anti-VEGF single domain antibodies according to the third aspect of the invention for use in preparations for tumor diagnosis;

In another preferred example, said use is diagnostic and/or non-diagnostic and/or therapeutic and/or non-therapeutic.

A twelfth aspect of the invention provides an antibody, said antibody comprising one or more VHH chains of an anti-VEGF single domain antibody according to the second aspect of the invention.

In another preferred example, said antibody comprises two VHH chains of an anti-VEGF single domain antibody according to the second aspect of the invention.

In another preferred example, said antibody has a heavy chain variable region VHH according to the second aspect of the invention.

In another preferred example, said antibody may be specific for a VEGFA protein with correct spatial conformation.

In another preferred embodiment, the antibody is capable of distinguishing between human, mouse, rabbit and monkey VEGFA.

In another preferred example, the antibody does not cross-react with human VEGFB, VEGFC, VEGFD.

In another preferred example, the antibody is capable of blocking interactions between VEGFA and VEGFR2 and VEGFA and VEGFR1.

In another preferred example, the antibody is capable of inhibiting the formation of angiogenesis.

In another preferred example, said antibody is a single domain antibody.

A thirteenth aspect of the invention provides a recombinant protein, said recombinant protein comprising:
(i) a VHH chain according to the second aspect of the invention or an anti-VEGF single domain antibody according to the third aspect of the invention, and (ii) an optional tag sequence that cooperates in expression and/or purification. have

In another preferred example, said tag sequence comprises Fc tag, HA tag and 6His tag.

In another preferred example, said recombinant protein specifically binds to VEGFA protein.

A fourteenth aspect of the invention is the VHH chain of the anti-VEGF single domain antibody according to the second aspect of the invention, the VHH chain of the third aspect of the invention, for use in the preparation of a medicament, reagent, detection plate or kit. providing the use of an anti-VEGF single domain antibody according to aspect or an immunoconjugate according to the eighth aspect of the invention;
wherein said reagent, detection plate or kit is used to detect VEGF protein in a sample;
wherein said medicament is used for the treatment or prevention of a disease or condition associated with VEGF.

In another preferred example, said detection includes flow detection, cell immunofluorescence detection.

In another preferred example, said disease or condition comprises a tumor or cancer or eye disease.

In another preferred example, the tumor or cancer comprises one or more of breast cancer, lung cancer, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, thyroid cancer, nasopharyngeal cancer, Not limited.

In another preferred example, the eye disease includes age-related macular degeneration, diabetic retinopathy, retinal vein occlusion, pathological myopia, neovascular glaucoma and other eye diseases involving neovascularization, including but not limited to: Not limited.

A fifteenth aspect of the invention provides a method of treating a disease, said method comprising administering to a subject in need thereof a single domain antibody according to the third aspect of the invention, or the eighth aspect of the invention. administering an immunoconjugate as described in .

In another preferred example, the subject includes mammals such as humans, mice, rabbit pups, and monkeys.

A sixteenth aspect of the invention provides a method of detecting VEGFA protein in a sample, said method comprising:
(1) contacting the sample with a VHH chain according to the second aspect of the invention, a single domain antibody according to the third aspect of the invention, or an immunoconjugate according to the eighth aspect of the invention; and
(2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of VEGFA protein in the sample.

In another preferred example, said method is a non-diagnostic and non-therapeutic method.

A seventeenth aspect of the invention provides a VEGFA protein detection reagent, said detection reagent comprising:
(i) a VHH chain according to the second aspect of the invention, a single domain antibody according to the third aspect of the invention, or an immunoconjugate according to the eighth aspect of the invention, and (ii) ) detectably acceptable vectors.

In another preferred example, the coupling moiety of said immunoconjugate is a diagnostic isotope.

In another preferred embodiment, the detectably acceptable vector is a non-toxic, inert aqueous vector vehicle.

In another preferred example, the detection reagent is one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow detection reagents, cellular immunofluorescence detection reagents, magnetic nanoparticles and imaging agents. is.

In another preferred example, said detection reagent is used for in vivo detection.

In another preferred example, the dosage form of the detection reagent is liquid or powder (eg, aqueous solution, injection, lyophilized powder, tablet, oral formulation, inhalant).

An eighteenth aspect of the invention provides a kit for detecting a VEGFA protein, said kit comprising an immunoconjugate according to the eighth aspect of the invention or a detection reagent according to the seventeenth aspect of the invention; and including instructions.

In another preferred example, the instructions state that the kit non-invasively detects VEGFA expression in the test subject.

A nineteenth aspect of the invention provides use of the immunoconjugate according to the eighth aspect of the invention for the preparation of an imaging agent for detecting VEGFA protein in vivo.

In another preferred example, said detection is used for cancer diagnosis or prognosis.

A twentieth aspect of the present invention provides a framework region FR of an anti-VEGF single domain antibody VHH chain, wherein the framework region FR of said VHH chain comprises SEQ ID NO. FR1 shown in :4, SEQ ID NO. : FR2, SEQ ID NO. FR3 shown in :6, SEQ ID NO. :7 or FR4 shown in SEQ ID NO. FR1 shown in :10, SEQ ID NO. FR2 shown in :11, SEQ ID NO. :12, and SEQ ID NO. : 13 is composed of FR4.

A twenty-first aspect of the invention provides a method of treating a disease or condition associated with VEGF comprising administering to a subject in need thereof the pharmaceutical composition of the tenth aspect of the invention.

In another preferred example, the subject comprises mammals such as humans.

Within the scope of the present invention, by combining each of the above technical features of the present invention with each of the technical features specifically described below (e.g., examples), new or preferable technical It should be appreciated that solutions can be constructed. Due to limited space, it will not be repeated here.

Figure 2 shows the results of ELISA identification of single domain antibodies capable of blocking the interaction between human VEGFA and VEGFR2. The blocking activity of the candidate antibody Nb24 is identified to be significantly superior to that of the control antibody Avastin. where Avastin is Bevacizumab. Figure 2 shows the results of detecting the inhibitory effect of candidate antibodies on HUVEC cell proliferation. The inhibitory effect of the candidate antibody Nb24 on HUVEC cell proliferation is identified to be stronger than that of the control antibody Avastin. SEC-HPLC detection results of huNb24 bivalent expressed in yeast are shown. A sample purified by ammonium sulfate precipitation was identified by SEC-HPLC, and the purity of the sample can reach 94.11%. ELISA detection of blocking activity of candidate antibodies after humanization. The results show that the humanization is successful as the blocking activity of the antibody before and after humanization is substantial (IC _{50 Nb24} =0.045 ug/mL, IC _{50 huNb24} =0.038 ug/mL). where Nb24 is the whole humanized antibody and huNb24 is the humanized antibody. ELISA detection of blocking activity of humanized bivalent antibodies expressed in yeast. The results show that the bivalent blocking activity of the single domain antibody expressed in yeast is significantly improved (IC _{50 huNb24} =0.044 ug/mL, IC _{50 hu bi-Nb24(Y)} =0.013 ug/mL). mL), indicating a significantly higher blocking activity than the control antibody Avastin (IC _{50 Avastin} = 0.331 ug/mL). Here, hubi-Nb24(Y) is a humanized bivalent antibody expressed in yeast. Comparison of blocking activity of humanized bivalent antibodies and similar commercially available products by ELISA detection. The results show that the blocking activity of the yeast-expressed humanized dimeric antibody has clear advantages and is superior to the blocking activity of some commercial products (IC _{50 hub bi-Nb24 (Y)} = 0.022 ug/mL, IC50 _Eylea = 0.085 ug/mL, IC50 _Conbercept = 0.088 ug/mL, IC50 _Avastin = 0.439 ug/mL). Here Eylea is Aflibercept and Conbercept is Conbercept. FIG. 10 is a detection result of the inhibitory effect of yeast-expressed humanized bivalent antibodies on HUVEC cellular proliferation. Results show that the efficacy of the yeast-expressed humanized bivalent antibody is superior to that of a similar commercial control product ( _{IC50hubi-Nb24(Y)} = 53.59 ng/mL, IC50 _Eylea = 65.96 ng/mL, IC50 _Conbercept = 129.7 ng/mL, IC50 _Avastin = 254.7 ng/mL). It is the result of detecting whether the candidate antibody can cross-react with the VEGF homologous protein by ELISA. The results show that the humanized bivalent antibody can react with human VEGFA without cross-reacting with other homologous proteins such as VEGFB, VEGFC, VEGFD, and its specificity is good. It is the result of detecting whether the candidate antibody can cross-react with other species of VEGF by ELISA. The results show that the humanized bivalent antibody can simultaneously discriminate between human, mouse and rabbit VEGFA. Blockade of yeast-expressed humanized bivalent antibodies detected by ELISA against the interaction between human VEGFA and VEGFR1. The results showed that the candidate antibody was able to block the interaction between human VEGFA and VEGFR1 ( _{IC50hubi-Nb24(Y)} = 0.168 ug/mL), with blocking activity higher than that of the control antibody Avastin ( _{IC50 Avastin} = 0.967 ug/mL). It is a statistical result of the area of the retinal non-perfused region in OIR model mice. Mice were administered with different concentrations of yeast-expressed humanized bivalent antibodies, and the area of the retinal non-perfused area of mice in the experimental group was smaller than that in the positive control group. Statistical results of retinal neovascularization clusters in OIR model mice. Compared to aflibercept Eylea (positive control), the humanized bivalent antibody expressed in different concentrations of yeast showed a more obvious inhibitory effect on retinal neovascular clusters in mice, with a statistical difference have SEC-HPLC was used to detect the stability of candidate antibodies at various temperatures. FIG. 13A shows the stability results of the candidate antibody at 4° C. for 1 month, the results showing that the antibody was kept at 4° C. under non-formulated conditions for 1 month, followed by a loss of purity. There was no obvious change, indicating good stability. FIG. 13B shows the stability results of the candidate antibody at 25° C. for 1 month, and the results showed that the antibody reached 25° C. in purity after 1 month at 25° C. under non-formulated conditions. There was no obvious change, indicating good stability. FIG. 13C shows the stability results of the candidate antibody at 40° C. for 15 days, showing a clear change in purity after the antibody was placed at 40° C. for 15 days under non-formulated conditions. , indicating good stability. FIG. 13D shows the stability results of the candidate antibody subjected to 5 cycles of freezing and thawing at −20° C., showing that after the antibody was placed at −20° C. under non-formulated conditions, 5 repeated freeze-thaw cycles with no apparent change in purity, indicating good stability.

The present inventors have unexpectedly discovered for the first time a class of anti-VEGF single domain antibodies after extensive detailed research and extensive screening, and experimental results show that the single domain antibodies of the present invention are , can specifically discriminate VEGFA without cross-reacting with VEGFB, VEGFC, VEGFD, has good specificity, and effectively recognizes the interaction between VEGFA and VEGFR2 and between VEGFA and VEGFR1. , indicating that it has a good inhibitory effect on angiogenesis. Generation of single domain antibodies of the invention is straightforward. Based on this, the present invention was completed.

Specifically, the present invention uses human VEGFA protein to immunize camels to obtain a high quality immune single domain antibody gene library. VEGFR protein molecules are then coupled to ELISA plates to demonstrate the correct spatial structure of the VEGFR protein, and this form of antigen can be used to screen immune single domain antibody gene libraries using bacteriophage display technology. (camelid heavy chain antibody bacteriophage display gene library) to obtain VEGFA-specific single domain antibody genes. Furthermore, by introducing this gene into mammalian cells, a single domain antibody strain that can be highly efficiently expressed in mammalian cells and has high specificity is obtained. Anti-VEGF single domain antibodies with blocking activity are then identified by methods such as ELISA, flow cytometry, luciferase reporter gene detection system, and the like.

Terms As used herein, "single domain antibody of the invention", "single domain antibody of the invention", "anti-VEGF antibody of the invention", "VEGF single domain antibody of the invention", The term "anti-VEGF single domain antibody" has the same meaning and is used interchangeably and both refer to single domain antibodies that specifically recognize and bind VEGFA (including human VEGFA). Preferably, the variable regions of the single domain antibodies of the invention have SEQ ID NO. CDR1 shown in :1, SEQ ID NO. :2, and SEQ ID NO. :3 with CDR3. More preferably, the framework regions of the single domain antibodies of the invention are (a) SEQ ID NO. FR1 shown in :4, SEQ ID NO. : FR2, SEQ ID NO. :6, and SEQ ID NO. :7, or (b) SEQ ID NO. FR1 shown in :10, SEQ ID NO. FR2 shown in :11, SEQ ID NO. :12, and SEQ ID NO. : 13 with FR4.

As used herein, the terms "antibody" or "immunoglobulin" have the same structural characteristics, composed of two identical light chains (L) and two identical heavy chains (H). It is a heterotetraglycan protein of approximately 150,000 daltons. Each light chain is joined to a heavy chain by a covalent disulfide bond, with different numbers of disulfide bonds between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. At one end of each heavy chain is a variable region (VH) followed by a number of constant regions. At one end of each light chain is a variable region (VL) and at the other end is a constant region; the constant region of the light chain is opposite the first constant region of the heavy chain; The region is opposite the variable region of the heavy chain. Specialized amino acid residues form an interface between the light and heavy chain variable regions.

As used herein, the terms "single domain antibody", "VHH", "nanobody", "single domain antibody" (single domain antibody, sdAb, or nanobody) are , which has the same meaning and is used interchangeably, cloning the variable region of an antibody heavy chain to form a single heavy chain variable region consisting of only one heavy chain variable region, which is the smallest fully functional antigen-binding fragment. Refers to constructing domain antibodies (VHHs). Usually, after obtaining an antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1), the variable region of the antibody heavy chain is cloned into a single antibody consisting of only one heavy chain variable region. A domain antibody (VHH) is constructed.

As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ in sequence that contribute to the binding and specificity of each particular antibody for its particular antigen. show. However, the variability is not evenly distributed throughout antibody variable regions. It is concentrated in three fragments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions in variable regions are called the framework regions (FR). Naturally occurring heavy and light chain variable regions each contain four FR regions, which are generally in a β-sheet configuration and connected by three CDRs that form a link loop, in certain cases partially A β-sheet structure can be formed. The CDRs of each chain are tightly bound by the FR regions and together with the CDRs of another chain form the antibody's antigen-binding site (Kabat et al., NIH Publ, No. 91-3242, Vol. I, pp. 647-669). (1991)). Although the constant regions are not directly involved in antibody-antigen binding, they exhibit various effector functions, including involvement in antibody-dependent cellular cytotoxicity of antibodies.

Immunoconjugates and fusion expression products, as known to those of skill in the art, include drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules combined with antibodies or fragments thereof of the present invention. including conjugates formed by binding to The invention further includes cell surface markers or antigens that bind to said anti-VEGF antibodies or fragments thereof.

As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably.

As used herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

In a preferred embodiment of the invention, the heavy chain variable region of said antibody comprises three complementarity determining regions CDR1, CDR2 and CDR3.

In a preferred embodiment of the invention, the heavy chain of said antibody comprises said heavy chain variable region and heavy chain constant region.

In the present invention, the terms "antibody of the invention", "protein of the invention", or "polypeptide of the invention" are used interchangeably and both refer to a polypeptide that specifically binds to a VEGF protein; For example, it refers to a protein or polypeptide having a heavy chain variable region. They may or may not contain an initiating methionine.

In general, the antigen-binding properties of an antibody can be described by three specific regions located in the heavy chain variable region, called variable regions (CDRs), which separate the segments into four framework regions (FRs). However, the amino acid sequences of the four FRs are relatively conserved and are not directly involved in binding reactions. These CDRs form a circular structure and are close to each other in a spatial configuration via β-sheets formed by FRs between them, and the CDRs of the heavy chain and the corresponding light chain CDRs form the antigen-binding site of the antibody. Configure. The amino acids that make up the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.

The variable regions of the heavy chains of the antibodies of the invention are of particular interest, as at least some of them are involved in antigen recognition. Accordingly, the present invention provides CDR-containing antibody heavy chain variable regions, so long as the CDRs have 90% or more (preferably 95% or more, most preferably 98% or more) homology with the CDRs identified herein. contains molecules that have

The present invention includes not only complete antibodies, but also fragments, derivatives and analogues of said antibodies.

As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that retain substantially the same biological function or activity as the antibody of the invention. A polypeptide fragment, derivative or analogue of the invention may comprise (i) one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) encoded or not encoded by the genetic code. or (ii) having substitutions at one or more amino acid residues, or (iii) the mature polypeptide and another compound (e.g., polyethylene glycol, etc.). or (iv) an additional amino acid sequence to this polypeptide sequence (e.g., a leader or secretory sequence or this polypeptide or a proprotein sequence, or a fusion protein formed with a 6His tag). These fragments, derivatives and analogs fall within the purview of those skilled in the art following the teachings of the present specification.

Antibodies of the present invention refer to polypeptides containing the above CDR regions that have VEGFA binding activity. The term further includes variants of the above CDR region-containing polypeptides that have the same function as the antibodies of the invention. These variants have one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, and most preferably 1 to 10) amino acid deletions and insertions. and/or substitutions, and the addition of one or more (usually no more than 20, preferably no more than 10, more preferably no more than 5) amino acids to the C-terminus and/or N-terminus. not limited). For example, substitutions made with amino acids of similar or similar performance in the art generally do not alter protein function. As another example, addition of one or more amino acids to the C-terminus and/or N-terminus generally does not alter protein function. The term further includes active fragments and derivatives of the antibodies of the invention.

Variants of the polypeptides include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA encoding the antibodies of the invention under conditions of high or low stringency. and polypeptides or proteins obtained using antisera against the antibodies of the invention.

The invention provides other polypeptides, and in addition to most full-length polypeptides, the invention further includes fragments of the single domain antibodies of the invention. Generally, such fragments will be at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, most preferably at least It has about 100 consecutive amino acids.

In the present invention, "conservative variants of the antibody of the present invention" refer to at most 10, preferably at most 8, more preferably at most 5, most preferably Refers to the replacement of up to three amino acids by amino acids of similar or similar properties to form a polypeptide. These conservative variant polypeptides are preferably generated by amino acid replacement according to Table 1.

本発明は、上記抗体またはそのフラグメントをコードするポリヌクレオチド分子をさらに提供する。本発明のポリヌクレオチドは、ＤＮＡ形態またはＲＮＡ形態であり得る。ＤＮＡ形態は、ｃＤＮＡ、ゲノムＤＮＡまたは人工的に合成したＤＮＡを含む。ＤＮＡは、一本鎖または二本鎖であり得る。ＤＮＡは、コード鎖または非コード鎖であり得る。

The invention further provides polynucleotide molecules encoding the above antibodies or fragments thereof. The polynucleotides of the invention can be in DNA or RNA form. DNA forms include cDNA, genomic DNA or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be the coding strand or the non-coding strand.

A polynucleotide encoding a mature polypeptide of the present invention includes a coding sequence encoding only the mature polypeptide, a mature polypeptide coding sequence and various additional coding sequences, and a mature polypeptide coding sequence (optionally additional coding sequences) and non-coding sequences.

The term "polynucleotide encoding a polypeptide" can be a polynucleotide encoding the polypeptide or a polynucleotide further comprising additional coding and/or non-coding sequences.

The invention further relates to polynucleotides that hybridize to the above sequences and have at least 50%, preferably at least 70%, more preferably at least 80% homology between the two sequences. The present invention particularly relates to polynucleotides that are hybridizable under stringent conditions to said polynucleotides of the invention. In the present invention, "stringent conditions" means (1) hybridization and elution under lower ionic strength and higher temperature such as 0.2 × SSC, 0.1% SDS, 60 ° C., or (2) for hybridization, the addition of a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll at 42°C; It refers to hybridization occurring only when the identity between the sequences is at least 90% or more, more preferably 95% or more. Also, a polypeptide encoded by a hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

The full-length nucleotide sequences of the antibodies of the present invention or fragments thereof can usually be obtained by PCR amplification methods, recombinant methods or artificial synthesis methods. A viable method is to use artificial synthesis to synthesize the tethering sequences, especially when the length of the fragments is short. Fragments of very long sequences can usually be obtained by first synthesizing multiple smaller fragments and then joining them. Additionally, the heavy chain coding sequence can be fused to an expression tag (eg, 6His) to form a fusion protein.

Once the related sequences are obtained, recombination methods can be used to obtain the related sequences en masse. This is usually cloned into a vector, transferred into cells and then isolated from the host cells grown by conventional methods to obtain the relevant sequences. Biomolecules (nucleic acids, proteins, etc.) referred to in the present invention include biomolecules that exist in isolated form.

DNA sequences encoding the proteins of the invention (or fragments thereof or derivatives thereof) can now be obtained entirely by chemical synthesis. The DNA sequences can then be introduced into a variety of existing DNA molecules (or vectors, etc.) and cells known in the art. Additionally, mutations can be introduced into the protein sequences of the invention by chemical synthesis.

The invention further relates to vectors containing the above suitable DNA sequences and suitable promoter or control sequences. These vectors can be used to transform suitable host cells so that the proteins can be expressed.

Host cells can be prokaryotic cells such as bacterial cells, or lower eukaryotic cells such as yeast cells, or higher eukaryotic cells such as mammalian cells. Typical examples include bacterial cells of Escherichia coli, Streptomyces, and Salmonella, Shinkin cells such as yeast, insect cells such as Drosophila S2 or Sf9, and animal cells such as CHO, COS7, and 293 cells.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those of skill in the art. If the host is a prokaryote such as E. coli, competent cells capable of assimilating DNA after the exponential growth phase can be harvested and treated with the _CaCl2 method using procedures well known in the art. . Another method is to use _MgCl2 . If desired, transformation can also be performed by electroporation. If the host is eukaryotic, DNA transfection methods such as calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc. can be used.

The resulting transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the invention. Depending on the host cell used, the medium used for culture can be selected from various conventional media. It is cultured under conditions suitable for growth of the host cells. After the host cells have grown to a suitable cell density, the selected promoter is induced using a suitable method (eg, temperature shift or chemical induction) and the cells are further cultured for a period of time.

The recombinant polypeptides in the above methods can be expressed intracellularly or on the cell membrane, or can be secreted extracellularly. As desired, its physical, chemical and other properties can be used to isolate and purify the recombinant protein by a variety of isolation methods. These methods are well known to those skilled in the art. Examples of these methods include conventional regeneration, treatment with protein precipitants (salting out), centrifugation, osmotic sterilization, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography. , ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

The antibodies of the invention can be used alone, or can be conjugated to detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties or combinations of any of these agents described above. can be coupled.

Detectable markers for diagnostic purposes include, but are not limited to, fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product. not.

Therapeutic agents that can be bound or coupled to the antibodies of the present invention include (1) radionuclides, (2) biotoxicity, (3) cytokines such as IL-2, (4) gold nanoparticles/nanorods, (5) virus particles (6) liposomes; (7) magnetic nanoparticles; (8) prodrug-activating enzymes (e.g. DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); (10) chemotherapeutic agents (e.g. , cisplatin) or any form of nanoparticles, etc.

Vascular Endothelial Growth Factor (VEGF)
Vascular Endothelial Growth Factor (VEGF) is a highly specific vascular endothelial cell growth factor and VEGF has a receptor for its receptor on the endothelial cell membrane, the Vascular Endothelial Growth Factor Receptor, VEGFR), causing autophosphorylation of the receptor, thereby activating mitogen-activated protein kinase (MAPK), achieving mitogenic properties and inducing endothelial cell proliferation. Due to its angiogenic properties, VEGF can restore oxygenation to tissues when blood circulation is inadequate. When VEGF is overexpressed in tissues, it can cause disease symptoms. For example, overexpression of VEGF can lead to retinal vascular diseases such as diabetic retinopathy. Furthermore, solid tumors cannot grow beyond a certain size limit without an adequate vascular supply to obtain the nutrients they need for growth. VEGF is expressed to allow growth and metastasis.

Members of the VEGF family include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and Placenta Growth Factor (PGF). Here, VEGF-A is the most important factor capable of regulating normal and pathological neovascularization (ie, angiogenesis). The biological effects of VEGF-A are mediated by binding to its specific receptors, primarily the specific receptors vascular endothelial growth factor receptor 1 (VEGFR-1) and the specific receptor binds to vascular endothelial growth factor receptor 2 (VEGFR-2). Here, VEGFR-2 is thought to be the major VEGFR that has a significant effect on the proliferation of vascular endothelial cells. VEGFR-2 enhances cellular mitogenesis by inducing VEGF to bind receptors requiring dimerization and autophosphorylation via intracellular kinases. VEGF-C and VEGF-D can regulate the formation of lymphatic vessels.

Pharmaceutical Compositions The present invention further provides compositions. Preferably, said composition is a pharmaceutical composition, which comprises said antibody or active fragment thereof, and a pharmaceutically acceptable vector. Generally, these substances can be prepared in a non-toxic, inert, pharmaceutically acceptable aqueous vector medium, where the pH value depends on the nature of the substance to be prepared and the disease to be treated. Although the pH can vary, the pH is usually about 5-8, preferably about 6-8. The prepared pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.

The pharmaceutical composition of the present invention can be used directly for conjugation of VEGFA protein molecules and thus can be used for the treatment of tumors. Additionally, other therapeutic agents can be used concurrently.

The pharmaceutical composition of the present invention contains a safe and effective amount (eg, 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described single domain antibody of the present invention. (or a conjugate thereof) and a pharmaceutically acceptable vector or excipient. Such vectors include, but are not limited to: saline, buffers, glucose, water, glycerol, ethanol, and combinations thereof. A pharmaceutical formulation should be compatible with the mode of administration. Pharmaceutical compositions of the present invention in the form of injections can be prepared by conventional methods, for example, using saline or aqueous solutions containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dose of active ingredient is a therapeutically effective amount, eg, from about 10 μg/kg body weight to about 50 mg/kg body weight per day. Additionally, the polypeptides of the invention can be used in conjunction with still other therapeutic agents.

When using pharmaceutical compositions, a safe and effective amount of the immunoconjugate is administered to a mammal, where the safe and effective amount is usually at least about 10 μg/kg body weight, and in most cases about 50 mg. /kg body weight or less, preferably the dose is about 10 μg/kg body weight to about 10 mg/kg body weight. Of course, the specific dosage should also take into account the route of administration, health condition of the patient, etc., all of which are within the skill of a skilled physician.

Anti-VEGF Single Domain Antibodies In the present invention, said anti-VEGF single domain antibodies include monomers, bivalents (bivalent antibodies) and/or multivalents (multivalent antibodies).

In a preferred embodiment of the invention, said anti-VEGF single domain antibody has SEQ ID NO. :8 and/or SEQ ID NO. :14.

Typically, said anti-VEGF single domain antibody has SEQ ID NO. :8 and/or SEQ ID NO. It contains two VHH chains with amino acid sequences as shown in :14.

Typically, said anti-VEGF single domain antibody has SEQ ID NO. :8 and/or SEQ ID NO. :14 with the amino acid sequence of the VHH chain.

Typically, said anti-VEGF single domain antibody has SEQ ID NO. It contains two VHH chains with amino acid sequences as shown in :14.

In a preferred embodiment of the invention, the SEQ ID NO. :8 are connected by a linker between two VHH chains having amino acid sequences as shown in FIG.

In a preferred embodiment of the invention, the SEQ ID NO. :14 are connected by a linker between the two VHH chains.

In a preferred embodiment of the invention, said linker is selected from the sequence (GaSb) _x- ( _GmSn ) _y , where a, _b , _m , _n , x, y=0 or 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 (preferably a=4 and b=1, m=3 and n=1).

In a preferred embodiment of the invention, said linker is selected from the group consisting of GGGGSGGGGS (SEQ ID NO.:18), GS (SEQ ID NO.:19), GGGGS (SEQ ID NO.:20).

In a preferred embodiment of the invention, the amino acid sequence of said anti-VEGF single domain antibody is SEQ ID NO. :16.

In a preferred embodiment of the invention said bivalent anti-VEGF single domain antibody is hubi-Nb24(Y).

Labeled Single Domain Antibodies In a preferred embodiment of the invention, said single domain antibodies carry a detectable marker. More preferably, said markers are selected from the group consisting of isotopes, colloidal gold markers, colored markers or fluorescent markers.

Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred form of the invention, the anti-VEGF single domain antibody is produced using a colloidal gold label to obtain a colloidal gold labeled single domain antibody.

The novel anti-VEGF single domain antibodies of the invention have excellent specificity and very high potency.

Detection Methods The present invention further relates to methods of detecting VEGF protein. The steps of the method are roughly as follows. A cell and/or tissue sample is obtained, the sample is lysed in a medium, and the level of VEGF protein is detected in the lysed sample.

The sample used in the detection method of the present invention is not particularly limited, and a typical example is a cell-containing sample present in a cell preservation solution.

Kits The present invention further provides kits comprising the antibodies (or fragments thereof) or detection plates of the present invention, and in preferred examples of the invention, said kits further comprise containers, instructions, buffers and the like.

The invention further provides a detection kit for detecting VEGF levels, the kit comprising an antibody that identifies the VEGF protein, a pyrolysis medium for lysing the sample, e.g., various buffers, a detection label, detection Contains common reagents and buffers required for detection of substrates, etc. The detection kit can be an in vitro diagnostic device.

Applications As mentioned above, the single domain antibody of the present invention has a wide range of biological and clinical application values, and its applications include diagnosis and treatment of VEGF-related diseases, basic medical research, biology Relating to many areas such as research. Preferred applications are clinical diagnostics and targeted therapy for VEGF.

The main advantages of the invention are:
(a) The single domain antibody specificity of the invention is protein to VEGF with the correct spatial structure.
(b) The single domain antibodies of the invention are capable of distinguishing between human, mouse, rabbit and monkey VEGF.
(c) The single domain antibody of the invention only recognizes human VEGFA, does not cross-react with VEGFB, VEGFC, VEGFD and has good specificity.
(d) The single domain antibodies of the invention are capable of blocking the interaction between VEGFA and VEGFR2 and between VEGFA and VEGFR1, the blocking activity being higher than that of aflibercept.
(e) The single domain antibody of the present invention has a good inhibitory effect on angiogenesis, and the effect is superior to that of the commercial product aflibercept.
(f) The single domain antibody of the present invention has good anti-tumor activity, and the efficacy is superior to that of the commercial product Avastin.
(g) The production of single domain antibodies of the invention is straightforward.
(h) Single domain antibodies of the invention exhibit better stability under non-formulated conditions.

The invention is further illustrated by the following specific examples. It should be understood that these examples are used only to illustrate the invention and are not intended to limit the scope of the invention. Experimental methods that do not indicate specific conditions in the following examples are generally based on general conditions, such as (Sambrook and Russell et al., Molecular Cloning-A Laboratory Manual, 3rd ed.) ( 2001) CSHL Publishers), according to the conditions suggested by the manufacturer. Percentages and parts are calculated by weight unless otherwise indicated.

Example 1 Screening and Expression of Anti-VEGF Single Domain Antibodies To obtain single domain antibodies specific for human VEGF, mammalian HEK293F cells were first used to transiently express human VEGFA protein. and used for camel immunization after affinity purification. For the specific method, refer to the method description in Example 1 and Example 2 of Patent CN2018101517526. Briefly, two Xinjiang Bactrian camels were immunized with purified VEGFA protein, total RNA was isolated from camel peripheral blood after 7 immunizations, and then VHH genes were amplified by reverse transcription and PCR. , the VHH gene is cloned into the bacteriophage vector pMECS and transformed into TG1 to construct a bacteriophage display library. The library capacities of the constructed libraries are 6.4×10 ⁸ CFU and 5.5×10 ⁸ CFU, respectively, and the insertion rates are 91.7% and 95.8%, respectively. After that, the libraries are screened, and the two libraries are screened for 6 and 5 rounds respectively to obtain an enrichment of bacteriophage containing antibody genes. 300 clones were selected from each library to perform PE-ELISA identification, the resulting positive clones were sequenced, and single domain antibodies of different sequences were fused to Fc and expressed using the HEK293F system. , the antibody is transiently expressed, and the expression method refers to the method description in Example 3 of patent CN2018101517526.

Example 2 Screening for Blocking Anti-VEGF Single Domain Antibodies An ELISA method is used to screen for single domain antibodies that can block the interaction between human VEGFA and VEGFR2. (1) VEGFR2 protein was coated on an ELISA plate (1 ug/mL, 100 uL/well) and incubated overnight at 4°C, (2) washed 5 times with PBST, and then added with 300 uL of 1% BSA blocking solution. , incubated at 37° C. for 2 hours, (3) washed 5 times with PBST, then added 50 uL of gradient diluted antibody sample (2-fold gradient dilution from 40 ug/mL), 50 uL of 0.08 ug to each well. /mL biotinylated VEGFA protein was added and incubated for 1 hour at 37°C, (4) after washing 5 times with PBST, 100 uL of SA-HRP (1:100000 dilution) was added and incubated for 1 hour at 37°C. (5) After washing 5 times with PBST, add 100 uL of TMB coloring solution, develop the color at 37° C. for 10 minutes, add 50 uL/well of 2M H ₂ SO ₄ to stop the reaction, and use a microplate reader. Absorbance is measured at a wavelength of 450 nm. The results show that the blocking activity of Nb24 is significantly superior to that of the control antibody Avastin (IC50 _Nb24 = 0.0149 ug/mL, IC50 _Avastin = 0.2172 ug/mL, as shown in Figure 1). ).

Example 3 Inhibitory Effect of Candidate Antibodies on HUVEC Cell Proliferation Briefly, the method is as follows. (1) Well-grown HUVEC cells were trypsinized, neutralized the entire medium, washed once with PBS, resuspended at a concentration of 3E4/mL, dispensed 100 uL/well into 96-well plates and placed at 37°C. Incubate at 5% CO ₂ for 20 hours. (2) The next day, dilute VEGFA to 100 ng/mL in DMEM with 2% FBS, antibody Gradient dilution to 39.06 ng/mL, 9.77 ng/mL, 2 ng/mL. (3) Add 60 uL of VEGFA and an equal amount of diluted positive antibody to another 96-well plate, mix evenly, co-culture at 37° C. for 2 hours, each mixture in 3 duplicate wells. (4) Remove the cell culture plate from the incubator, aspirate the supernatant, then add 100ul of the mixture (3) to each corresponding well and incubate at 37°C for 72 hours. (5) After 72 hours, add 10 ul/well of CCK8 solution, develop color for 2 hours, and read the absorbance value at OD450 wavelength with a microplate reader after completion of color development. The results show that the inhibitory effect of the candidate antibody Nb24 on HUVEC cell proliferation is stronger than that of the control antibody Avastin (IC50 _Nb24 = 29.58 ng/mL, IC50 Avastin = 72.58 ng/mL, IC50 _Avastin = 72.58 ng/mL). 28 ng/mL).

Example 4: Antibody Nb24 Humanization and Expression Humanizing the candidate antibody without altering the variable region, performing the humanization design on the sequences of the four framework regions, the alteration method is described in Example 4 of patent CN2018101517526 See how. The humanized antibody huNb24 sequence was constructed into a pFUSE vector to fuse the humanized single domain antibody with the Fc sequence and expressed using the HEK293F system, and the protein after expression was used for subsequent validation. can be The modified sequences are as shown in Table 2 below.

Example 5 Construction and Expression of a Humanized Bivalent Antibody The above humanized antibody was constructed in a bivalent form and ligated using the linker GGGGSGGGGS (SEQ ID NO.: 18), the amino acid sequence after ligation being: SEQ ID NO. :16 (the corresponding coding nucleotide sequence thereof is shown in SEQ ID NO.:17) and is subsequently expressed by Pichia pastoris. Briefly, the expression method is as follows. (1) SEQ ID NO. :16 was constructed into the pPICZaA vector, and (2) pPICZaA-Nb24-Nb24 was linearized with Sac I restriction endonuclease and then electrophoresed into X-33 competent cells. (3) the electrotransformed samples were each coated onto YPD plate media containing various concentrations of bleomycin resistance and cultured in an incubator at 30° C. for 3-4 days; You can refer to the pPICZaA vector instructions provided by the company; When the OD value of the culture reaches about 20, the cells are replaced with BMMY medium after collection, cultured at 28° C. and 250 rpm, (5) sampling every 24 hours thereafter, and the final volume is 1%. is added for sampling, the sample is centrifuged at 12000 rpm for 5 minutes, the supernatant is collected, stored under −20° C., continuously induced for 5 days, the culture is terminated, (6) collection The collected samples are detected by SDS-PAGE, at the same time samples are collected and the target antibody is purified by ammonium sulfate precipitation. The results are as shown in FIG. 3, the single domain antibody bivalent antibody hubi-Nb24(Y) purified by ammonium sulfate precipitation from the expression supernatant was detected by SEC-HPLC and its purity was , 94.11% and can be used for subsequent studies.

Example 6 Target Blocking Activity of Humanized Antibodies and Bivalent Antibodies by ELISA Detection The detection method was the same as in Example 2, and the results are shown in FIG. The blocking activity was similar to that of the antibody prior to humanization (IC _{50 Nb24} =0.045 ug/mL, IC _{50 huNb24} =0.038 ug/mL), thus indicating that this humanization modification was successful. Comparing the blocking activity of the humanized single domain antibody and the yeast expressed bivalent antibody, the above experiment was repeated and the results show that the yeast expressed single domain antibody The blocking activity of the bivalent hu bi-Nb24(Y) was increased more than 3-fold (IC 50 hu Bi- _Nb24 = 0.044 ug/mL, IC _{50 hu bi-Nb24(Y)} = 0.013 ug/mL) compared to the control It was significantly higher than the blocking activity of antibody Avastin (IC _{50 Avastin} = 0.331 ug/mL). The experimental methods described above were used to detect and compare the blocking activity of candidate antibodies with similar commercially available products, and the results are shown in FIG. - The blocking activity of Nb24(Y) has a clear advantage ( _{IC50hu bi-Nb24(Y)} = 0.022ug/mL, IC50 _Eylea = 0.085ug/mL, IC50 _convercept = 0.088ug /mL, IC50 _Avastin = 0.439 ug/mL). This indicates that the yeast-expressed humanized dimer has significantly better blocking activity than existing commercial products.

Example 7 Inhibitory Effect of Humanized Bivalent Antibodies on HUVEC Cell Proliferation The detection method was the same as in Example 3, and the results were expressed in yeast on HUVEC cell proliferation, as shown in FIG. The inhibitory effect of humanized bivalent antibody hubi-Nb24(Y) is superior to similar commercial control products (IC50 _hubi-Nb24(Y) = 53.59 ng/mL, IC50 _Eylea = 65.96 ng /mL, IC50 _Conbercept = 129.7 ng/mL, IC50 _Avastin = 254.7 ng/mL).

Example 8 Specificity of Candidate Antibodies by ELISA Detection ELISA is used to verify whether candidate antibodies can cross-react with VEGF homologous proteins. (1) Coat 1 ug/mL of test antibody to 100 uL/well ELISA plate overnight at 4°C, (2) Wash 5 times with PBST, then add 300 uL of 1% BSA to each well, Block for 2 hours at room temperature, (3) wash 5 times with PBST, then add 100 uL of 1 ug/mL biotin (Biotin)-hVEGFA, biotin-hVEGFB, biotin-hVEGFC, biotin-hVEGFD, and place at 37°C. Incubate for 1 hour, (4) wash 5 times with PBST, add 100 uL of diluted SA-HRP (1:5000 dilution), incubate at 37° C. for 1 hour, (5) wash 5 times with PBST. After that, add 100 uL of TMB coloring solution, develop the color at 37° C. for 10 minutes, add 50 uL/well of 2M H ₂ SO ₄ to stop the reaction, and measure the absorbance at a wavelength of 450 nm with a microplate reader. . The results show that the humanized bivalent antibody hubi-Nb24(Y) can react with human VEGFA without cross-reacting with other homologous proteins such as VEGFB, VEGFC, VEGFD, etc., as shown in FIG. can.

Similarly, ELISAs are used to detect whether a candidate antibody is capable of cross-reacting with VEGF from other species. (1) Coat 1 ug/mL of test antibody to 100 uL/well ELISA plate overnight at 4°C, (2) Wash 5 times with PBST, then add 300 uL of 1% BSA to each well, After blocking for 2 hours at room temperature, (3) washing with PBST 5 times, 100 uL of 1 ug/mL biotin-hVEGFA (human), biotin-mVEGFA (mouse), biotin-rVEGFA (rabbit) was added and incubated at 37°C. (4) washed 5 times with PBST, added 100 uL of diluted SA-HRP (1:5000 dilution), incubated for 1 hour at 37°C, (5) washed 5 times with PBST. After washing twice, 100 uL of TMB coloring solution was added to develop the color at 37° C. for 10 minutes, 50 uL/well of 2M H ₂ SO ₄ was added to stop the reaction, and the absorbance was measured at a wavelength of 450 nm using a microplate reader. Measure. The results show that the humanized bivalent antibody hubi-Nb24(Y) can simultaneously discriminate human, mouse and rabbit VEGFA, as shown in FIG. Furthermore, since the sequences at the corresponding positions in human VEGF121 and crab-eating monkeys are exactly the same, it is believed that the candidate antibodies can also discriminate crab-eating monkey VEGFAs.

Example 9: Blocking activity of humanized bivalent antibodies against VEGFR1/VEGFA by ELISA detection (1) VEGFR1 protein was coated on an ELISA plate (1 ug/mL, 100 uL/well), incubated overnight at 4°C, ( 2) After washing 5 times with PBST, add 300 uL of 1% BSA blocking solution and incubate at 37°C for 2 hours; (3) Wash 5 times with PBST, then add 50 uL of gradient diluted antibody samples 2-fold gradient dilution from 20 ug/mL), 50 uL of 0.08 ug/mL biotinylated VEGFA protein was added to each well, incubated at 37° C. for 1 hour, (4) washed 5 times with PBST, followed by 100 uL of Add SA-HRP (1:100000 dilution), incubate at 37° C. for 1 hour, (5) wash with PBST 5 times, add 100 uL of TMB coloring solution, develop color at 37° C. for 10 minutes, and add 50 uL. 2M H ₂ SO ₄ /well is added to stop the reaction and the absorbance is measured under a wavelength of 450 nm in a microplate reader. The results show that the candidate antibody hubi-Nb24(Y) can block the interaction between human VEGFA and VEGFR1 (IC50 _hubi-Nb24(Y) = 0 .168 ug/mL), the blocking activity is superior to that of the control antibody Avastin (IC _{50 Avastin} = 0.967 ug/mL).

Example 10: Inhibitory Efficacy of Candidate Antibodies on Intraocular Blood Vessel Growth in Neonatal Mouse OIR Model (1) A classical mouse model for studying angiogenesis—OIR model is used. Seven-day-old male C57BL/6J mice were housed in closed containers with 75% oxygen concentration for 12 days, during which time the oxygen concentration in the oxygen box was regularly monitored and maintained at 75%, followed by Raise for 5 days under a normal air environment. P7 (7 days old)-P12 (12 days old) corresponds to the retinal vessel occlusion period, P12-P17 (17 days old) corresponds to the hypoxia-abnormal vessel proliferation period, P17-P21 ( 21 days of age) corresponds to a period of abnormally proliferating vessel recovery. P17 was selected as the observation point for abnormal angiogenesis, and for P12, 1 uL of antibody drug hubi-Nb24 (Y) of different concentrations was injected into the vitreous of mice in the OIR model group (1.5 mg/mL, 1.0 mg/mL, 0.5 mg/mL), the concentration of the control antibody Eylea is 40 mg/mL, then the mice are transferred to normal air and housed until P17.
(2) Staining FITC-IB4 retinal vascular endothelial cells for retinal plating and observing abnormal neovascular clusters and retinal non-perfused areas for P17 in the OIR model: after sacrificing C57BL/6J mice. , Immediately remove the mouse eyeball, fix it with 4% paraformaldehyde for 1-2 hours, cut the spherical surface along the corneal limbus under an operating microscope, remove the lens and vitreous body, and remove the retinal nerve fiber layer. and pigment epithelium, remove sclera, choroid, retinal pigment epithelium layer, rinse retinal nerve fiber layer with PBS, remove residual vitreous, containing 1 mg/mL BSA and 0.3% TX-100 Block with PBS for 1-2 hours, add FITC-IB4 solution (1:50 dilution) for 48 hours at 4°C, remove, rinse with PBS for 15 minutes x 3 times, and plate the retinal nerve fiber layer on a glass slide. The rats were then cut, cut radially around the optic nerve head, mounted with mounting medium, observed and photographed under fluorescence and confocal microscopes, and statisticed.

The results are shown in Figures 11 and 12, in Figure 11, three concentrations of the drug hubi-Nb24(Y) (1.5 mg/mL, 1.0 mg/mL) compared to the OIR negative control group. mL, 0.5 mg/mL) was able to reduce the area of the non-perfused retinal area and was statistically significant (p<0.05), compared with the positive control drug aflibercept Eylea (40 mg/mL). mL) can also reduce the area of the retinal non-perfused area. The areas of the retinal non-perfused areas at three concentrations (1.5 mg/mL, 1.0 mg/mL, and 0.5 mg/mL) of the drug hu bi-Nb24(Y) were all lower than the areas of the Eylea retinal non-perfused areas. are small, but not statistically different, compared to the OIR control in FIG. /mL) and Eylea were all able to reduce angiogenic clusters, all with statistical significance, compared with Eylea at three concentrations of the drug hu bi-Nb24(Y) (1.5 mg/mL). mL, 1.0 mg/mL, 0.5 mg/mL), the percentages of retinal neovascular clusters were all small and had statistical differences, and with increasing concentrations of the drug hubi-Nb24(Y), P17 The percentage of retinal neovascular clusters decreases when ).

Example 11: Candidate Antibody Stability Study Candidate antibody hu bi-Nb24(Y) at a concentration of 1 mg/mL under the conditions of -20°C (repeated freeze-thaw), 4°C, 25°C, and 40°C, respectively. Place in 10 mM PB solution and take samples at different time points for SEC-HPLC detection. Using Advance Bio SEC 130A 2.7um 7.8*300mm column, detection wavelength is 280nm, tassel is 0.5mL/min at room temperature, moving 200mM pH7.0PB solution The phases are isocratically eluted for 30 minutes.

As shown in FIG. 13, the detection results were as follows: 4° C. for 1 month (FIG. 13A), 25° C. for 1 month (FIG. 13B), 40° C. for 15 days (FIG. 13C), −20° C. There was no apparent change in the purity of the candidate antibody under five freeze-thaw cycles (Fig. 13D), indicating that the antibody exhibits better stability under non-formulated conditions.

All documents mentioned in this application are incorporated by reference in this application as if each document were individually incorporated by reference. Moreover, after reading the above teachings of the invention, one skilled in the art may make various changes or modifications to the invention, the equivalents of which are also within the scope defined by the appended claims of this application. include.

Claims (16)

A complementarity determining region (CDR) of an anti-VEGF single domain antibody VHH chain, comprising:
The complementarity determining region CDRs of said VHH chains are identified by SEQ ID NO. CDR1 shown in :1, SEQ ID NO. :2, and SEQ ID NO. : the complementarity determining region (CDR) of said anti-VEGF single domain antibody VHH chain, characterized in that it comprises the CDR3 shown in 3. A VHH chain of an anti-VEGF single domain antibody,
Said VHH chain comprises a framework region FR and a complementarity determining region CDR according to claim 1, preferably said framework region FR comprises
(a) SEQ ID NO. FR1 shown in :4, SEQ ID NO. : FR2, SEQ ID NO. :6, and SEQ ID NO. :7, or (b) SEQ ID NO. FR1 shown in :10, SEQ ID NO. FR2 shown in :11, SEQ ID NO. : 12, and SEQ ID NO. : VHH chain of said anti-VEGF single domain antibody, characterized in that it comprises FR4 shown in 13. an anti-VEGF antibody,
Said anti-VEGF antibody, characterized in that it has the VHH chain according to claim 2. Said anti-VEGF antibody has SEQ ID NO. : 8 or SEQ ID NO. : comprising one or more VHH chains having amino acid sequences as shown in
The anti-VEGF antibody of claim 3. The amino acid sequence of said anti-VEGF antibody is shown in SEQ ID NO. : 16, characterized in that
The anti-VEGF antibody of claim 3. a polynucleotide,
The polynucleotide consists of the CDR region of the anti-VEGF single domain antibody VHH chain of claim 1, the VHH chain of the anti-VEGF single domain antibody of claim 2, and the anti-VEGF antibody of claim 3. Said polynucleotide, characterized in that it encodes a protein selected from the group. an expression vector,
7. The expression vector, wherein the expression vector comprises the polynucleotide of claim 6. a host cell,
Said host cell, characterized in that it contains the expression vector of claim 7 or has the polynucleotide of claim 6 integrated in its genome. A method of producing an anti-VEGF single domain antibody comprising:
(a) obtaining a culture comprising said anti-VEGF single domain antibody by culturing the host cell of claim 8 under conditions suitable for the production of single domain antibodies;
(b) isolating or recovering said anti-VEGF single domain antibody from said culture; and (c) optionally purifying and/or modifying to obtain the VEGF single domain antibody of step (b). A method of producing said anti-VEGF single domain antibody, comprising: an immunoconjugate,
The immunoconjugate is
(a) the VHH chain of the anti-VEGF single domain antibody of claim 2, or the anti-VEGF antibody of claim 3; and (b) a detectable marker, drug, toxin, cytokine, radionuclide, enzyme. , gold nanoparticles/nanorods, magnetic nanoparticles, viral coat proteins or VLPs, or a combination thereof. Use of the VHH chain of the anti-VEGF single domain antibody according to claim 2 or the anti-VEGF antibody according to claim 3,
(a) a drug for inhibiting angiogenesis;
(b) Said use, characterized in that it is used in the preparation of a medicament for treating a disease or condition associated with VEGF. A pharmaceutical composition comprising
The pharmaceutical composition comprises
(a) the complementarity determining region (CDR) of the anti-VEGF single domain antibody VHH chain of claim 1, the VHH chain of the anti-VEGF single domain antibody of claim 2, or the anti-VEGF single domain antibody of claim 3 11. Said pharmaceutical composition comprising a VEGF antibody, or the immunoconjugate of claim 10, and (b) a pharmaceutically acceptable vector. an antibody,
3. An antibody, characterized in that it comprises one or more VHH chains of the anti-VEGF single domain antibody of claim 2. a recombinant protein,
The recombinant protein is
(i) the VHH chain of claim 2 or the anti-VEGF antibody of claim 3; and (ii) an optional tag sequence that cooperates in expression and/or purification. recombinant protein. A method of detecting VEGFA protein in a sample comprising:
The method includes:
(1) contacting the sample with the VHH chain of claim 2, the antibody of claim 3 or the immunoconjugate of claim 10;
(2) detecting whether antigen-antibody complexes are formed, wherein the formation of complexes indicates the presence of VEGFA protein in the sample. A method for detecting VEGFA protein in. A VEGFA protein detection reagent comprising:
The detection reagent is
(i) the VHH chain of claim 2, the antibody of claim 3, or the immunoconjugate of claim 10; and (ii) a detectably acceptable vector. and the VEGFA protein detection reagent.

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